Rotation 1: Investigating the role(s) of the putative FANCJ helicase in the malaria parasite Plasmodium falciparum

第 1 轮：研究假定的 FANCJ 解旋酶在疟原虫恶性疟原虫中的作用

• 批准号: 2886902
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2886902
• 关键词: Rotation; Investigating; role; putative; FANCJ

Bioscience for an integrated understanding of healthThe airways of people with cystic fibrosis (CF) are often colonised by a diverse "zoo" of microbes, especially during their early and teenage years. These polymicrobial communities have historically proven difficult to recapitulate in the laboratory environment, so, until now, most research has involved monoculture of key pathogens such as Pseudomonas aeruginosa. It is becoming increasingly clear, however, that pathogens in multi-species systems often behave radically differently than when grown in pure axenic cultures, and, as such, recent years have seen a growing interest in efforts to recapitulate the complex CF microbiota in vitro. The Welch Lab has developed an in vitro laboratory model that accurately captures the long-term stability of the polymicrobial community present in CF airways. In brief, the setup consists of a continuous flow bioreactor in which the rate of fluid - specifically, artificial sputum media - replacement reflects that seen in the airways. The current model incorporates three major CF-associated pathogens: P. aeruginosa, Staphylococcus aureus, and Candida albicans. This system has opened new possibilities for interrogation of the biology of CF-associated polymicrobial airway infections, and has already yielded some intriguing new insights. In this project, I will introduce other common CF-associated pathogens into the in vitro model system, alongside the current three, for example: Haemophilus influenzae, Rothia mucilaginosa, and Streptococcus milleri. I will then use this "improved" model system to examine the impacts of the presence of P. aeruginosa mutants in the polymicrobial community. The P. aeruginosa population observed in CF airway infections is not homogenous, with mutant variants frequently observed. Loss-of-function mutations in certain genes are commonly over-represented in CF, indicating they may confer increased fitness in this environment; however, we still have very little idea as to why this is. Key "pathoadaptive" genes include mutS (mutations leading to hypermutability), lasR (affecting virulence quorum sensing), mucA (mucoidy), mexT (ciprofloxacin resistance), and metF (methionine auxotrophy). To investigate the impact of these pathoadaptive mutations on P. aeruginosa fitness, I will generate loss-of-function mutants for each target gene, then examine how introduction of these mutants (or mixtures of both wild type and mutant(s)) influences the diversity and species trajectory of the polymicrobial culture. This will further our understanding of how the arrival of P. aeruginosa into the CF airway influences the existing microbiome, and whether some mutants are more successful at colonising this niche than others. Preliminary data from the Welch Group indicate that lasR mutants do indeed show majorly altered inter-species interactions as compared to the wild type, and I hope to see similarly interesting results for other pathoadaptive mutants. I will also examine how the presence of pathoadaptive mutants in the model system impacts clinically relevant factors, such as response of the involved pathogens to antimicrobials. This project will help us understand why some mutants arise with such high frequency in CF, the advantages these mutants potentially confer on P. aeruginosa, and their impact on therapeutic interventions: it therefore has both biological and clinical interest and importance.

生物科学对健康的综合了解，囊性纤维化（CF）的气道通常被微生物的“动物园”殖民，尤其是在他们的早期和青少年时期。这些多数群落在历史上被证明很难在实验室环境中概括，因此，到目前为止，大多数研究涉及诸如铜绿假单胞菌等关键病原体的单一文化。然而，越来越清楚的是，多物种系统中的病原体通常与在纯轴突培养物中生长时的行为通常不同，因此，近年来，人们对在体外概括复杂的CF Microbobiota的努力越来越兴趣。 Welch Lab开发了一种体外实验室模型，该模型可以准确捕获CF Airways中存在多数菌群社区的长期稳定性。简而言之，该设置由连续的流动生物反应器组成，其中流体速率（特别是人造痰介质）的替换反映了在气道中看到的。当前模型结合了三种主要的CF相关病原体：铜绿假单胞菌，金黄色葡萄球菌和白色念珠菌。该系统为询问与CF相关的多数气道感染的生物学开辟了新的可能性，并且已经产生了一些有趣的新见解。在这个项目中，我将与当前三个相关的CF相关病原体引入体外模型系统，例如：流感嗜血杆菌，Rothia Mucilaginosa和Milleri链球菌。然后，我将使用此“改进”模型系统来检查多因素群落中铜绿假单胞菌突变体存在的影响。在CF气道感染中观察到的铜绿假单胞菌种群不是均匀的，突变体变体经常观察到。某些基因的功能丧失突变通常在CF中代表过多，表明它们可能会在这种环境下提高适合度；但是，我们对为什么这样的想法几乎没有任何想法。关键的“致病自适应”基因包括MUTS（突变导致过敏性），LASR（影响毒力群体传感），粘膜（粘液胶质），Mext（环丙沙星抗性）和METF（甲硫酸丙氨酸）。为了研究这些病原体突变对铜绿假单胞菌适应性的影响，我将为每个靶基因产生功能丧失的突变体，然后检查这些突变体（或野生型和突变体的混合物（或混合物）的引入如何影响多因素培养物的多样性和物种轨迹。这将进一步了解我们对铜绿假单胞菌进入CF气道的到来如何影响现有的微生物组，以及某些突变体是否比其他突变体更成功地殖民这种利基。来自韦尔奇组的初步数据表明，与野生型相比，LASR突变体确实确实显示出主要变化的种间相互作用，我希望能看到其他病原适应性突变体的有趣结果。我还将研究模型系统中病原体突变体的存在如何影响临床相关的因素，例如所涉及的病原体对抗菌剂的反应。该项目将帮助我们理解为什么某些突变体在CF中如此高频率，这些突变体可能会赋予铜绿假单胞菌的优势及其对治疗干预措施的影响：因此，它具有生物学和临床的兴趣和重要性。